Study of human aging processes is important, in part because many diseases or conditions become more prominent among aged people, for example, cancers, Alzheimer's disease, Parkinson's disease, stroke, heart failure, and heart attack, just to name a few, among which many still lack effective preventive or treatment methods. Therefore, the search for more effective prevention or treatment methods for the age-related diseases or disorders through studies of animal aging processes has become one of the most important endeavors embarked by the scientific community in the last decade. Although abundant literature has contributed to the understanding of aging processes, full understanding of the processes remains to be a major scientific challenge faced by mankind. Given the increasing population of aged people around the world and the rising health care burden and cost associated therewith, systematic studies of the aging processes leading to effective discovery of anti-aging agents for the prevention or treatment of age-related diseases or disorders are becoming increasingly important. This invention represents such a systematic approach aiming to provide effective methods for the discovery of anti-aging agents that can be developed into effective prevention and/or treatment of age-related diseases or disorders.
Among various theories concerning the aging processes and the methods derived from the theories that could be useful for the treatment of age-related diseases, the nutrient signaling pathway (caloric restriction), the mitochondria pathway (reactive oxygen species, or ROS), and the telomere dysfunction theory are prominent.
Nutrient Signaling Pathway (Caloric Restriction) and Aging. Caloric restriction (CR) has been recognized as the most practicable method to retard the rate of aging from yeast to mammals. CR has also been shown to reduce the incidence or delay the onset of age-related diseases such as Parkinson's disease in a primate model (Maswood, N., et al., Proc. Natl. Acad. Sci. USA, 101:18171-6 (2004)), Alzheimer's disease (Qin, W., et al., J. Alzheimer's Dis., 10:417-422 (2006)), hypertension and heart problems in the Dahl-SS rat model (Seymour, E. M., et al., J. Mol. Cell Cardiol., 41:661-668 (2006)), fibrosis (Castello, L., et al., FASEB J., 19:1863-1865 (2005)), and kidney disease (Yu, B. P., et al., J. Gerontol., 37:130-141 (1982)). CR also inhibits a variety of spontaneous neoplasias and decreases the incident of human breast, colon and prostate cancers (reviewed in Platz, E. A., J. Nutr., 132:3471S-81S (2002); Steinbach, G., et al., Cancer Res., 54:1194-1197 (1994); Michels, K. B., et al., JAMA, 291:1226-30 (2004)).
The well-conserved kinase TOR (Target of rapamycin) integrates signals from nutrients, mitogenic growth factors, energy, and stress to regulate catabolic and anabolic processes (Fingar, D. C., et al., Oncogene, 23:3151-3171 (2004)). In response to optimal growth factors and nutrients, mammalian TOR (mTOR) stimulates the cell's synthetic capabilities (such as ribosome biogenesis and protein translation initiation), leading to increases in cell mass and size and accelerates proliferation (Kim, E., et al., Hum. Gene Ther., 14:1415-1428 (2003)). Conversely, inhibition of TOR by growth factor withdrawal, nutrient starvation, or stress leads to the down-regulation of high energy-consuming processes and inhibition of proliferation.
TOR pathway may play an important role in the life span extension induced by CR in budding yeast, Ceanorhabditis elegans and Drosophila (Kaeberlein, M., et al., Science, 310:1193-1196 (2005); Powers, R. W., et al., Genes Dev., 20:174-84 (2006); Vellai, T., et al., Nature, 426:620 (2003); Kapahi, P., et al., Curr. Biol., 14:885-890 (2004); Jia, K., et al., Development, 131:3897-3906 (2004)). As the function of TOR is well conserved, its role in aging may also apply to humans.
The Mitochondrion/ROS and Aging. Mitochondria are cellular organelles responsible for converting metabolic fuels (e.g., glucose and fatty acids) into a usable form of energy, adenosine 5′-triphosphate (ATP), through the process of oxidative phosphorylation. Mitochondria are also involved in other processes that are important for proper cellular function, including calcium homeostasis, intracellular signal transduction, and the regulation of apoptosis.
The process of oxidative phosphorylation for ATP generation in mitochondria is also the main source of reactive oxygen species (ROS) within the cell (about 90% of total ROS in cells) (Balaban, R. S., et al., Cell, 120:483-495 (2005)). Under normal physiological conditions, ROS leaked during oxidative phosphorylation is estimated to represent 1-5% of the oxygen consumed during this process (Chance, B., et al., Physiol. Rev., 59:527-605 (1979)). Due to the limited repair capacity of mitochondrial DNA (mtDNA) and the proximity to the oxidants, mitochondria are particularly vulnerable to accumulation of damages. Mutations in mtDNA then result in impaired function of oxidative phosphorylation, leading to increased ROS production and the subsequent accumulation of more mutations. As ROS are highly reactive molecules and can generate diverse damages in the cells, the ROS vicious cycle is believed to account for an exponential increase in oxidative damage during aging, which results in a gradually functional decline that characterizes the aging process.
ROS may be associated with many age-related diseases, for example, diabetes, cardiovascular disease, cancer and Parkinson's disease (Kovacic, P., et al., Curr. Med. Chem., 8:773-796 (2001); Aviram, M., et al., Am. J. Clin. Nutr., 71:1062-1076 (2000); Maassen, J. A., et al., J. Endocrinol. Invest., 25:477-484 (2002)). The fact that eukaryotes develop a host anti-oxidant defense system also supports the important role of endogenous ROS production (Mates, J. M., Toxicology, 153:83-104 (2000)) and overexpression of superoxide dismutase and catalase extends life span in Drosophila melanogaster (Orr, W. C., et al., Science, 263:1128-1130 (1994)).
Previous studies indicate that mitochondrial integrity declines as a function of age as monitored by decreases in mitochondrial membrane potential, mitochondrial number, and ATP generation/O2 consumption (Hagen, T. M., et al., Proc. Natl. Acad. Sci. USA, 94:3064-3069, 1997; Greco, M., et al., FASEB J., 17:1706-1708 (2003)). Mutations in mitochondrial function cause a number of human genetic diseases with clinical manifestations including blindness, deafness, movement disorders, dementias, cardiovascular disease, muscle weakness, renal dysfunction, and endocrine disorders. Furthermore, it has been reported that mice with a dramatic increase in mitochondrial DNA mutations (due to a proof-reading-deficient mutation in mtDNA polymerase PolgA) exhibited a shorter life span, accompanied with certain premature aging phenotypes (Trifunovic, A., et al., Nature, 429:417-423 (2004)). Moreover, life span extension in yeast (Chronological life span) by deletion of TOR1 and in C. Elegans by glucose restriction has been reported to be via mitochondrial respiration (Bonawitz, N. D., et al., Cell Metab., 5:233-235 (2007); Schulz, T. J., et al., Cell Metab., 6:280-293 (2007)). These results suggest the important role of mitochondrial function in aging and age-related diseases in mammals. However, contradictory results have also been reported. For example, CR-induced life span extension in budding yeast was reported to be independent of mitochondrial function (Kaeberlein, M., et al., PloS Genet., 1, e69 (2005)). Therefore, the role of mitochondria in the aging process remains to be unclear.
Telomeres, Senescence, Aging and Cancer. Telomeres are ends of chromosomes consisting of G-rich repeated DNA sequences on one strand. The telomeres are bound by telomere binding proteins to protect them from being recognized as the naturally occurring double-stranded DNA breaks (DSBs).
Dysfunctional telomeres can be caused by progressive shortening of telomeres due to the internal problem of DNA replication by DNA polymerase and the lack of telomerase activity in most somatic cells in humans. Eventually, the critically shortened telomeres cannot be bound by telomere proteins and are thus exposed as natural DSBs, which activate DNA damage responses and induce RB- and p53-dependent cell cycle arrest. This process is termed replicative senescence. In addition, senescence can be induced by oncogenes activation via the same DNA damage responses, resulting in tumor suppression (Di Micco, R., et al., Nature, 444:638-642 (2006); Bartkova, J., et al., Nature, 444:633-637 (2006)). Furthermore, DNA damage agents have also been reported to trigger senescence as well.
Telomere dysfunction also occurs when there is a telomere binding protein defect. For example, expression of a dominant-negative TTAGGG repeat binding factor 2 (TRF2), as well as knockdown of protection of telomeres 1 (POT1), results in telomere dysfunction and DNA damage signals (Karlseder, J., et al., Science, 283:1321-1325 (1999); Denchi, E. L., et al., Nature, 448:1068-1071 (2007); Guo, X., et al., EMBO J., 26:4709-4719 (2007)).
Long telomeres have been associated with longevity in humans, while short telomeres have been associated with cancers, idiopathic pulmonary fibrosis and a variety of proliferative tissue disorders. For example, the telomerase mutation in human causes Dyskeratosis congenita and patients typically die early of bone marrow failure.
Replication senescence has been shown to be a barrier for tumor progression, since cancer cells require unlimited replication potential. Indeed, senescent markers are prominent in pre-malignant lesions but undetectable in advanced cancers in mouse models and in human cancers (Braig, M., et al., Nature, 436:660-665 (2005); Collado, M., et al., Nature 436:642 (2005); Michaloglou, C., et al., Nature, 436:720-724 (2005)). All cancers bypass senescence by activating telomerase or alternative telomere lengthening by recombination (Shay, J. W., et al., Exp. Cell. Res., 209:45-52 (1993); Shay, J. W., et al., Eur. J. Cancer, 33:787-791 (1997); Kim, N. W., et al., Science, 266:2011-2015 (1994); Bryan, T. M., et al., Nat. Med., 3:271-274 (1997)). The progression of early stage prostate cancer to malignance is blocked by senescence (Chen, Z., et al., Nature, 436:725-730 (2005)). Furthermore, the spontaneous tumorigenesis induced by telomere dysfunction in telomerase mutant mice Terc−/− was shown to be inhibited by p53-mediated senescence (Cosme-Blanco, W., et al., EMBO Rep. 8:497-503 (2007)). Senescence is assumed to stop the cell cycle and facilitate repair, thus blocking further development of the initial lesions.
Senescence is also viewed as a major contributor to aging (Campisi, J., Nat. Rev. Cancer., 3:339-49; Faragher, R. G., Biochem. Soc. Trans., 28:221-226 (2000)). For example, senescent cells increase with age in mammalian tissues (Campisi, J., Cell, 120:1-10 (2005)). Senescent cells have been found at sites of age-related pathologies such as osteoarthritis and atherosclerosis (Price, J. S., et al., Aging Cell, 1:57-65 (2002); Vasile E., et al., FASEB J., 15:458-466 (2001); Matthews, C., et al., Cir. Res., 99:156-164 (2006)). Moreover, chronically active p53 both promotes cellular senescence and accelerates aging phenotypes in mice (Maier, B., et al., Genes Dev., 18: 306-319 (2004); Tyner et al., Nature, 415:45-53 (2002)). Furthermore, it has been shown that senescent cells secret proteins that facilitate tumor progression and inflammation response (Coppe, J. P., et al., PlosBiology, 6:2853-2868 (2008)). It has been proposed that the programmed senescence leads to age-related diseases and limited our life span (Blagosklonny, M. V., Cell Cycle, 5:2087-2102 (2006)). Therefore, anti-aging research from the telomere angle is currently focused on preventing senescence.
Despite all the studies, the role of telomeres in aging process remains unclear. For example, it cannot explain why a mouse has longer telomeres but a shorter life span than a human. It is not clear if or how telomeres work in the aging process of post-mitotic cells.
Other aging theories have also been proposed, for example, the protein damage accumulation theory, the DNA mutation accumulation theory, and the stem cell exhausting theory. Among the above, which theories represent the true nature of the aging processes and whether and/or how they are related to each other are still unclear. Therefore, at least to some degree, human aging processes remain to be a mystery.
Age-related diseases or disorders, such as cancers, cardiovascular diseases, and neuronal degeneration diseases, are leading causes of death in humans. Pharmaceutical agents for the treatment of these age-related diseases or disorders are being searched according to the current understanding of the specific diseases, due to the limited understanding about the aging processes. As a result, to date, these diseases have been studied independently of each other and disconnected from the aging processes. Therefore, there is a need to develop a systemic approach based on the aging processes to the discovery of novel anti-aging agents for the prevention and treatment of age-related diseases or disorders.